Evaluation of an indirect fluorescent antibody test for detecting Babesia argentina infection in cattle.
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Acute Disease↗
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Calf ligated intestinal segment test to detect enterotoxigenic Escherichia coli.
The ligated intestinal segment test in the young calf was utilized to establish the enterotoxigenicity of approximately 600 Escherichia coli isolates obtained from fecal specimens intestinal contents of calves with diarrheal disease. One hundred and fifty isolates were routinely tested in a single calf. There was little problem with false-positive reactions. False-negative reactions normally occurred only if the isolate was tested in the posterior 3 m of the small intestine. The ligated small intestine of the calf was distended by E. coli isolates of bovine, porcine, and human origin. Use of the calf ligated intestinal segment test is recommended to determine the enterotoxigenicity of E. coli isolates of bovine origin.
Animals↗
Line blot and western blot immunoassays for diagnosis of Mediterranean spotted fever.
The line blot, a new immunoassay in which antigens are placed on nitrocellulose as narrow lines, was evaluated for its sensitivity and specificity relative to the microimmunofluorescence assay for the diagnosis of Mediterranean spotted fever (MSF). The line blot assay was only slightly less sensitive and less specific than the microimmunofluorescence assay for detection of immunoglobulin M (IgM) or IgG in 100 serum specimens from 42 patients with MSF. No line blot reactions were observed among 50 control serum specimens from febrile patients with other illnesses. The line blot assay was largely group reactive for spotted fever rickettsiae, but 26% of the positive serum specimens also cross-reacted by IgM with Rickettsia typhi. Western immunoblotting was used to characterize the antigenic components recognized by 19 MSF serum specimens. For both IgM and IgG, lipopolysaccharide was the cross-reactive group antigen, whereas the high-molecular-weight species-specific protein antigens (SPAs) were the only reactive proteins. Relative to the other nine rickettsiae, Rickettsia bellii was unique both in exhibiting no SPA reactions and in having a lipopolysaccharide with a predominantly high-molecular-weight distribution. Although most of the 19 MSF serum specimens examined by Western blotting exhibited preferential reactivity to SPAs of two strains of R. conorii and weaker reactions to the other rickettsiae, 2 serum specimens exhibited SPA reactions consistent with typhus infections. In comparison with other assays, the line blot and Western blot immunoassays have advantages which may permit an improvement in the general availability and commercialization of assays for the serodiagnosis of rickettsial infections.
Antibodies, Bacterial↗
Comparison of four enzyme immunoassays with a western blot assay for the determination of type-specific antibodies to herpes simplex virus.
Most current enzyme immunoassays (EIAs) differentiate inadequately between types 1 and 2 herpes simplex virus (HSV) antibodies since significant cross-reactivity exists. We compared 4 IgG type-specific EIAs using a Western blot assay for resolution of discrepant results. The Diamedix had sensitivities of 100% for types 1 and 2 but specificities of only 71% and 61%, respectively. The cross-reactivity rate was 82% in positive samples tested. For HSV types 1 and 2, the Zeus sensitivities were 92% and 98%, respectively; specificities were 72% and 79%, respectively; the cross-reactivity rate was 54%. For HSV types 1 and 2, the Wampole sensitivities were 98% and 95%, respectively; specificities were 68% and 85%, respectively; the cross-reactivity rate was 47%. For HSV types 1 and 2, the Meridian sensitivities were 98% and 90%, respectively; specificities were 96% and 100%, respectively; no cross-reactivity was found between positive samples tested. While the Diamedix, Zeus, and Wampole assays showed good sensitivity, they lacked type specificity. The Meridian EIA offers the highest specificity along with no observed cross-reactivity. This EIA may be an easier, reliable alternative to Western blot for the determination of HSV type-specific antibodies.
Antibodies, Viral↗
[Antibody monstration in blastomyces infections].
The evidence of humoral antibodies against yeasts can be a valuable help in the diagnosis of such infections. It is however necessary to use several sensitive methods, which complete one another in the detection of the different immunoglobulins. In the interpretation of the results the titer dynamic is decisive, especially in those patients whose antibody answer is suppressed by their disease and/or their therapeutic treatment. In most of the yeast infections Candida albicans can be used as antigen for the detection of antibodies because there is a strong relationship between the antigens of most of the yeasts important in human infections. C. krusei, C. pseudotropicalis and C. guilliermondii do not display enough common antigens to make a diagnosis against these yeasts possible with C. albicans. In that case homologous antigens have to be used. Cross reactions with other microorganisms are less frequent than usually supposed. Therefore so called false positive Candida titers have to be examined very carefully by clinical signs and culture methods.
Antibodies, Fungal↗
Immunoglobulins for immunofluorescence.
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Antibodies↗
Validity of the allergy skin test.
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Allergens↗
Detection of Fasciola hepatica in infected intermediate hosts using RT-PCR.
Fasciola hepatica, the common bile duct fluke, is an economically important parasite of domestic livestock. Current research interest is directed toward an understanding of the parasite's biology at the intermediate host level. To permit study of seasonal transmission patterns and parasite/intermediate host interactions, a fasciolid-specific assay has been developed to detect infected snail vectors. This assay uses the reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify specifically a region of F. hepatica small-subunit rRNA, followed by hybridization to an F. hepatica-specific probe. The assay does not cross-react with 2 trematodes outside of the Fasciolidae but does detect Fascioloides magna rRNA. Sequence alignment with additional small-subunit rRNAs shows Fasciolopsis buski would also cross-react with the assay. The detection limit of the assay is 10 fg of fluke total RNA with 5 micrograms of snail RNA added as background. Additionally, the assay detects individual infected snails immediately after miracidial exposure and throughout the parasite's development period.
Animals↗
[Humoral immune response in humans and animals infected and immunized with Mycobacteria of the MAIS complex].
Hyperimmunization of mice and rabbits with surface (whole cells) and complex (ultrasound disintegrants) with the MAIS complex Mycobacterium antigens induced a humoral immune response which was largely aimed against cross-reacting than "unique" Mycobacterium antigens. Infection of BALB/c mice and rabbits with M. avium induced a humoral immune response which was chiefly against cross-antigenic Mycobacterium determinants. A series of consecutive immunochemical procedures were used to isolate from M. avium antiserum the antibody reacting with the antigenic determinants 18, 20, 24, 27, 39, 68, 94, kDa. Mab to the antigenic determinant 79 kDa of M. avium, which belong to the IgG class and unreacting with antigens of other Mycobacterium types were obtained. The antibodies isolated from hyperimmune serum and Mabs were used in the competitive EIA to determine antimycobacterial antibodies in man, mice, and rabbits. With Mab, positive results were obtained in 75% of M. avium-infected mice, 63.6% of rabbits and 63.6% of patients with M. avium infection (with 5.0% false positive results in humans). With antiserum, 83.3% of positive results were obtained in mice (with 10.0% of false positive results), 81.8 in rabbits (with 25% of false positive results) and 90.9% in M. avium-infected persons (with 30% of false positive results).
Animals↗
Enzyme immunoassay validation for qualitative detection of cocaine in sweat.
A solid-phase enzyme immunoassay (EIA) involving microtiter plates was modified for analysis of cocaine in sweat. Sweat was collected with the PharmChek sweat patch and drugs were eluted from the collection pad of the patch. The sweat contained primarily parent cocaine. The assay was determined to have cross-reactivity for cocaine of 102% relative to 100% for the benzoylecgonine (BE) calibrators and for cocaethylene of 148%. The optimum cutoff concentration for this modified assay, determined by receiver-operating characteristic curve analysis, was 10 micrograms/L cocaine or BE equivalents. At this concentration the assay had 94.5% sensitivity and 99.1% specificity vs gas chromatography-mass spectrometry (GC-MS) as an acceptable indicator of the true clinical state. The positive predictive value at a prevalence of 50% was 99%. Threshold analysis for positives suggested that the 95% confidence interval for a positive result by the EIA was between 12.5 and 15 micrograms/L and that quality-control samples at 5 and 15 micrograms/L could be run with each batch to certify the precision around the cutoff. All positive samples must be confirmed by GC-MS. The sensitivity and specificity of the overall analysis system (immunoassay screen and GC-MS confirmation) was 86% and 97%, with known cocaine dosing of volunteers as the acceptable indicator of the true clinical state.
Cocaine↗
Clinical issues associated with urine testing of substances of abuse.
Several factors may affect the validity and outcome of urine testing for abused drugs such as amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, ethanol, opiates, and phencyclidine. Urine is used for large-scale testing because acquisition of the sample is noninvasive and because most abused drugs can be detected in urine for a reasonable duration after ingestion. Urine testing for drugs of abuse is a two-step process. In the first step, screening assays are used to identify presumably positive specimens. Common screening tests are radioimmunoassays, enzyme immunoassays, fluorescence polarization immunoassay, and thin layer chromatography. Since they may be subject to cross-reactivity, once a possible positive sample has been identified by a preliminary test, a second more specific methodology, gas chromatography with mass spectrometry, is done to confirm the results. Knowledge of the pharmacology and pharmacokinetics of abused drugs affects selection and interpretation of test results.
Cross Reactions↗
Direct testing of blood cultures for detection of streptococcal antigens.
A direct, rapid, and simple method for the detection of streptococcal antigens of Lancefield groups A, B, C, D, and G from blood cultures was developed by using a coagglutination test. Fifty-five clinical specimens and 117 simulated blood cultures containing gram-positive cocci were tested. Out of 6,261 clinical blood cultures screened, 55 cultures from 53 patients were positive, with organisms resembling streptococci, by Gram stain. Of these cultures, 78% (43 of 55) were pure cultures of streptococci, and 22% (12 of 55) were mixed with at least one other organism. Of the 43 pure cultures only, correct reactions were obtained (grouping correctly or giving no cross-reactions, or both) with 86% (37 of 43) of the isolates, 12% (5 of 43) exhibited cross-reactions, and 2% (1 of 43) gave false-negative reactions. All of the cross-reacting isolates were Streptococcus pneumoniae, which reacted with the group C reagent, and the false-negative reaction occurred with a Streptococcus bovis isolate. However, by using a direct modified bile solubility test, the correct identification of the S. pneumoniae isolates was obtained. Therefore, by using the modified bile solubility test in conjunction with the direct grouping method, 98% (42 of 43) of the isolates in pure culture could be identified accurately and rapidly after the detection of a positive Gram stain. Correct grouping reactions were obtained with 83% (10 of 12) of the mixed blood cultures, and false-negative results occurred with 17% (2 of 12) of them. Both cultures contained an enterococcus and a gram-negative rod. Of the 117 simulated blood cultures, there was only one incorrect grouping reaction; this occurred with an S. bovis isolate that cross-reacted with the group C reagent. The direct grouping reaction was positive when blood cultures contained a minimum of 1 x 10(8) to 8 x 10(8) colony-forming units per ml. In general, this procedure provided information on the identification of the organism 24 h earlier than by conventional identification methods.
Animals↗
Halothane testing for malignant hyperthermia in swine: dose-response effects.
Purebred Pietrain malignant hyperthermia (MH)-susceptible pigs (n = 102) were subjected to halothane (0%, 1%, 2%, 3%, 4%, and 5%) in oxygen. The number of pigs in each group exhibiting muscle rigidity (MH(+) reaction) and the reaction times were recorded, as were the number of deaths resulting from MH. Mortality was not affected by the halothane concentration. However, halothane concentration did markedly affect the number of MH(+) reactions and the reaction times. False-negative reactions were apparent in the pigs at halothane concentrations less than 3%. Increasing the halothane concentration incrementally to 5% (from 0%) significantly (P less than 0.05) decreased reaction times between treatment groups. The reductions in reaction times which occurred in the pigs given the 3%, 4%, and 5% halothane concentrations (62.1, 56.2, and 50.05)--although significant (P less than 0.05)--would indicate that 3% halothane would generally be sufficient for MH testing.
Animals↗
A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples.
Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.
Bacteriological Techniques↗
[Tuberculin screening tests. Open comparative trial of Tine-Test and Monotest versus Mantoux 1:1000 (10 TU) in 119 outpatients].
The two percutaneous tuberculin tests Tine test (TT) (old tuberculin) and Monotest (MT) (PPD-tuberculin) were compared simultaneously with the standard intradermal reaction (Mantoux [MX] 1:1000 = 10 EPPD-RT23-tuberculin) in 119 unselected 16-81 year old outpatients (general medicine) in an open trial. The tests were read after 48 and/or 72 h. Indurations of greater than or equal to 10 mm (MX), greater than or equal to 2 mm (MT) and greater than or equal to 5 mm of one papule or confluescent papules (TT) were considered to be positive. 6-9 mm for MX and 2-4 mm for TT were considered doubtful. The overall agreement with MX (69.7% positive, 27.7% negative reactions) was 92.4% for MT and 68.1% for TT. Regarding sensitivity (false negative reactions), MT provided far better results than TT (8.4% vs. 44.6%). TT performed equally well when the doubtful reactions were reclassified as positive (8.4% false negative). It is recommended that the limit for a positive TT be lowered to greater than or equal to 2 mm, as other authors have suggested. MT always produced easily palpable and measurable indurations, whereas the borderline TT reactions (2-4 mm) were difficult to read and the size of the four papules varied considerably in most instances. Only MT showed significantly more positive readings after 72 h compared to 48 h. MT was better tolerated subjectively than TT with regard to application and skin reaction. The Monotest can be recommended as tuberculin screening test for general practice.
Adolescent↗
Comparison of methods for rapid immunofluroescent staining of group A streptococci and Neisseria gonorrhoeae.
Immunofluorescent staining reactions for group A streptococci and Neisseria gonorrhoeae were equivalent to those of standard "slow" methods when slides were heated on a serological water bath at 50 C for periods one to four minutes. No false negative reactions occurred when this method was used. False negative reactions occasionally occurred when previously described rapid methods which employed a slide warmer were used.
Fluorescent Antibody Technique↗
Relative effectiveness of selective plating agars for recovery of Salmonella species from selected high-moisture foods.
The relative effectiveness of 6 selective plating media were compared for effectiveness in recovery of Salmonella spp. from selected high-moisture foods. Three new plating agars (EF-18, Rambach, and xylose lysine Tergitol-4) and 3 selective plating agars (bismuth sulfite, Hektoen enteric, and xylose lysine desoxycholate) recommended by AOAC INTERNATIONAL and the Bacteriological Analytical Manual (BAM) were compared. The agars were streaked from cultures selectively enriched in selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium. The high-moisture foods studied were naturally contaminated pork sausage, chicken parts, turkey parts, and frog legs and artificially contaminated shrimp, oysters, egg yolks, and lettuce. The relative effectiveness of each selective plating agar was determined by recovery of Salmonella spp. and enumeration of false-positive and false-negative reactions. Although the new selective plating agars compared favorably with the AOAC/BAM-recommended agars, they offered no advantage. Incubation of selective enrichment broths at elevated temperatures decreased the numbers of false-positive and false-negative reactions for all 6 selective plating agars.
Agar↗
Antigen specific lymphocyte transformation induced by secreted antigens from Toxoplasma gondii.
Secreted (TSA) and water lysed (WLA) antigens derived from cell culture of the RH strain of Toxoplasma gondii have been used to induce antigen specific mitogenesis of lymphocytes from patients with symptomatic and asymptomatic toxoplasmosis. Lymphocyte responsiveness to WLA was similar to previous reports, with about 50% of patients showing a false negative reaction. Responses to TSA however were highly specific, with no false negative reactions. This increased specificity was not due to an increased response against TSA by patients' lymphocytes (P less than 0.001), but a lower TSA response by uninfected subjects' lymphocytes (P greater than 0.1) compared with WLA in both cases. In a minority of both infected and uninfected subjects, there was a low but detectable response to antigens secreted by the host cell line (HCA), and this was directly compared to their responses against TSA. There was at least a 10-fold increase in the patients' responses to TSA when compared with HCA (P less than 0.001), whereas there was no significant difference between the uninfected subjects' responses to these antigens (P greater than 0.1). Preliminary observations have suggested that TSA is distinct from other defined secreted antigens as both heat treatment and solid phase immunosorption did not have any noticeable effect on TSA-induced mitogenesis.
Adolescent↗